Recombinant protein expression represents a large portion of the production of proteins used in molecular biology, agronomy, veterinary science or medicine. For example, recombinant therapeutic antibodies represent a large percentage of biopharmaceuticals (i.e. medical drugs produced using biotechnology) produced.
Protein signal sequences, also called topogenic signals or signal peptides, play a central role in the targeting and translocation of nearly all secreted proteins and many integral membrane proteins in both prokaryotes and eukaryotes. The signal peptides from various proteins generally consist of three structurally, and possibly functionally distinct, regions: (1) an amino terminal (N-terminal) positively charged N-region (or N-domain), (2) a central hydrophobic H-region (or H-domain), and (3) a neutral but polar carboxy terminal (C-region or C-domain).
The determination of protein signal sequences is important to improve excretion and secretion of antibodies produced using an expression system such as bacteria, plants, and animals to produce effective drugs (especially therapeutic proteins). By adding a specific tag to an antibody of interest, it is possible to improve the antibody excretion or secretion in the extracellular environment. In this manner, the antibody is easier to harvest and is less likely to be degraded or to induce toxicity of the expression system by accumulating in the intracellular environment of the expression system. Thus, an antibody may be expressed as a fusion protein comprising a preferred N-terminal sequence fused to a mature sequence of the heavy chain or the light chain of an antibody.
To effectively use this technique, the signal peptides must be identified. However, the amino acid sequence of most recombinant therapeutic antibodies disclosed in publicly available databases does not include the sequences of the signal peptides. Furthermore, the signal peptide information required for production in mammalian cell culture is also absent in the case of monoclonal antibodies isolated through methods such as phage display.
Rituxan is the only antibody for which heavy and light chain signal peptide sequence information is available in public database. However, the availability of the information does not entail that the original signal peptide is optimized for the secretion of the antibody in the desired host cell selected for production.
Another issue associated with production of recombinant antibodies comprising a signal peptide is the cleavage heterogeneity. Cleavage heterogeneity may arise from nonspecific cleavage of a signal peptide by a signal peptidase. As cleavage of the signal peptide occurs within the variable region in the N-terminus of both the heavy and light chains, non-specific cleavage may affect antigen recognition.
Recombinant IgG production may also be affected by glycan heterogeneity that is present at the N-glycosylation site of the CH2 constant domain of the heavy chains. One recent study explored an LC/MS with a column-switching system to rapidly evaluate glycan heterogeneity.
Thus, there is a need to provide further signal peptides to create recombinant antibodies that are efficiently secreted and produced in expression systems.